The neural crest is a transient tissue of the vertebrate embryo that originates in the neural folds, invades the embryo, and differentiates in distinct locations into a wide array of adult cell types and tissues. Neural crest derivatives include neurons, Schwann cells, and glia of the autonomic and enteric nervous systems, most primary sensory neurons, endocrine cells (e.g., the adrenal medulla and C-cells of the thyroid), the smooth musculature of the cardiac outflow tract and great vessels, pigment cells of the skin and internal organs, tooth papillae, meninges, as well as bone, cartilage, and connective tissue (dermis) of the face, forehead and ventral part of the neck (LeDouarin and Kalcheim, The Neural Crest, Cambridge University Press, 1999).
Recently, Anderson et al. reported methods for the isolation and clonal propagation of mammalian neural crest stem cells. Their methods use a separation and culturing regimen and bioassays for establishing the generation of neural crest stem cell derivatives. In their method, the neural crest stem cells are cultured on a mixed substrate of poly-D-lysine and fibronectin to generate neurons and glia. The cells were also found to express low-affinity nerve growth factor receptor (LNGFR) and nestin, but do not express glial fibrillary acidic protein (GFAP) (See U.S. Pat. No. 5,589,376). One of the problems with this method is that the cells originate from the embryonic neural tube. The procedure raises ethical considerations, as several week old embryos would be destroyed during isolation of embryonic neural crest cells. Ethical issues aside, the method from a practical standpoint is not feasible, as it is unlikely to provide a sufficient amount of neural tissue to meet the demands of therapeutic procedures, such as transplantation for cell replacement therapy. Thus, this cell source is not particularly desirable for transplantation.
Alternatively, Miller et al. reported the isolation of multipotent neural stem cells (MNSCs) from the peripheral tissue of postnatal mammals, including juvenile and adult mammals. They identified skin as a source of MNSCs and set forth methods to purify skin-derived MNSCs, thus simplifying the harvesting of cells for transplantation relative to previous methods (See U.S. Pat. No. 6,787,355). However, this population of cells is still fairly heterogenous (i.e., at least 30% of the cells are multipotent stem cells), requiring a series of additional purification steps to become useful for human medical procedures. In particular, MNSCs require multiple sub-cultures over a period of several weeks during which time their stem cell characteristics could change through, for example, the action of autocrine or paracrine differentiation factors due to the close proximity of cells. Furthermore, the ontological source of the MNSCs is unclear.
Accordingly, it would be useful to have a method for producing a substantially homogenous population of neural crest stem cells from a readily accessible source for medical research and therapeutic purposes.